Process for producing N-substituted polyhydroxy nitrogen-containing heterocycles

ABSTRACT

A process for producing N-substituted amino compounds, a process for oxidizing an N-substituted amino compound with a microbe, or cell fragment or cell free extract thereof, and a process for oxidizing an N-substituted amino compound with a microbe, or cell fragment or cell free extract thereof and reducing the oxidized N-substituted amino compound to N-substituted-polyhydroxy piperidines based on N-substituted mannosamines, allosamines and altrosamines, N-substituted polyhydroxy pyrrolidines, and N-substituted polyhydroxy azetidines. In addition, a one pot process for producing N-substituted-polyhydroxy piperidines based on N-substituted mannosamines, allosamines and altrosamines, N-substituted polyhydroxy pyrrolidines, and N-substituted polyhydroxy azetidines from the respective sugar is disclosed. A second embodiment comprises novel compositions of N-substituted-amino-6-deoxy-2-ketohexuloses based on mannose, allose and altrose, N-substituted-amino-5-deoxy-2-ketopentuloses and 4-(N-substituted)-amino-1,3-dihydroxy-2-butanones.

This is a DIVISION, of U.S. Ser. No. 07/851,818 filed on Mar. 16, 1992,now U.S. Pat. No. 5,401,645.

BACKGROUND OF THE INVENTION

This invention relates to a process for production of N-substitutedpolyhydroxy nitrogen-containing heterocycles and intermediates for theirproduction. In one aspect, this invention relates to a process forproduction of N-substituted polyhydroxy piperidines based onN-substituted mannosamines, allosamines and altrosamines, N-substitutedpolyhydroxy pyrrolidines, N-substituted polyhydroxy azetidines andintermediates for their production.

A process for the preparation of 1-deoxynojirimycin in which1-amino-1-deoxyglucitol is oxidized microbiologically to give6-aminosorbose, which is then hydrogenated with a catalyst to give1-deoxynojirimycin is disclosed in U.S. Pat. No. 4,246,345. However, theyields of this process, in particular the low volume yields in themicrobiological reaction are related to degradation problems and shortreaction times, in addition no process for production of N-substitutedderivatives of 1-deoxynojirimycin is disclosed.

It is known that N-substituted derivatives of 1-deoxynojirimycin can bemade by protecting aminosorbitols with protecting groups which arestable in subsequent microbial oxidations. The protecting groups cansubsequently be removed by catalytic hydrogenation. Such a process isdisclosed in U.S. Pat. No. 4,266,025. In the '025 patent, protectedamino sugars are oxidized microbiologically to give protected6-aminosorboses, which are then isolated. The protective group is thenremoved by catalytic hydrogenation and the ring is reclosed to form theN-substituted derivatives of 1-deoxynojirimycin. However, the '025process is a complex process with multiple reaction steps and requires alarge amount of catalyst in the hydrogenation step. In addition, theunprotected 6-aminosorboses cannot be isolated as such.

U.S. Pat. No. 4,405,714 discloses a process for producing N-substitutedderivatives of 1-deoxynojirimycin in which glucose is converted into a1-amino-1-deoxyglucitol. The 1-amino-1-deoxyglucitol is then protectedby a protecting group which is stable in the subsequent microbiologicaloxidation process. The protecting group can then be removed under acidconditions. The compounds are oxidized microbially to give a protected6-aminosorbose. The protective group on the 6-aminosorbose is thenremoved under acid conditions. The 6-aminosorbose salt thus obtained ishydrogenated with a catalyst to give the N-substituted derivative of1-deoxynojirimycin. The '714 process, like the '025 process, is amultistep process which requires the use of protective groups to obtainN-substituted derivatives of 1-deoxynojirimycin.

It has been discovered that N-substituted derivatives of polyhydroxypiperidines based on N-substituted derivatives of mannosamine,allosamine and altrosamine, N-substituted derivatives of polyhydroxypyrrolidines, and N-substituted derivatives of polyhydroxy azetidinescan be made by a process which does not require the use of protectinggroups.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a process for preparing thecompounds of the invention which does not require the use of protectinggroups. It is a further object of the invention to provide an efficientand economic process for preparing the compounds of the invention whichis commercially viable.

According to the invention, a process is provided which comprisesoxidizing a compound selected from the group consisting ofN-substituted-1-deoxy-1-hexosamines based on mannose, allose andaltrose, N-substituted-1-deoxy-1-pentosamines,N-substituted-1-deoxy-1-tetrosamines, and salts thereof, with a microbeselected from the group consisting of bacteria of the familyAcetobacteraceae, bacteria of the genus Corynebacterium, and cellfragments or cell free extracts thereof, and producing a correspondingcompound selected from the group consisting ofN-substituted-amino-6-deoxy-2-ketohexuloses based on mannose, allose andaltrose, N-substituted-amino-5-deoxy-2-ketopentuloses,4-(N-substituted)-amino-1,3-dihydroxy-2-butanones, and salts thereof. Inone embodiment of the invention, the oxidized product is reduced toproduce a compound selected from the group consisting of N-substitutedpolyhydroxy piperidines based on N-substituted mannosamines, allosaminesand altrosamines, N-substituted polyhydroxy pyrrolidines, N-substitutedpolyhydroxy azetidines, and salts thereof. In a further embodiment ofthe invention, the material to be oxidized is produced by the aminationof a sugar selected from the group consisting of mannose, allose,altrose, ribose, arabinose and erythrose. In a still further embodimentof the invention, a process is provided which comprises converting asugar selected from the group consisting of mannose, allose, altrose,ribose, arabinose and erythrose to the corresponding reduced productselected from the group consisting of N-substituted polyhydroxypiperidines based on N-substituted mannosamines, allosamines andaltrosamines, N-substituted polyhydroxy pyrrolidines, N-substitutedpolyhydroxy azetidines, and salts thereof.

Further according to the invention, novel compositions ofN-substituted-amino-6-deoxy-2-ketohexuloses based on mannose, allose andaltrose, N-substituted-amino-5-deoxy-2-ketopentuloses and4-(N-substituted)-amino-1,3-dihydroxy-2-butanones are provided.

DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention relates to a process for oxidizing acompound selected from the group consisting ofN-substituted-1-deoxy-1-hexosamines based on mannose, allose andaltrose, N-substituted-1-deoxy-1-pentosamines,N-substituted-1-deoxy-1-tetrosamines and salts thereof, with a microbeselected from the group consisting of bacteria of the familyAcetobacteraceae, bacteria of the genus Corynebacterium, and cellfragments or cell free extracts thereof, and producing a correspondingcompound selected from the group consisting ofN-substituted-amino-6-deoxy-2-ketohexuloses based on mannose, allose andaltrose, N-substituted-amino-5-deoxy-2-ketopentuloses,4-(N-substituted)-amino-1,3-dihydroxy-2-butanones, and salts thereof.

A second embodiment of the invention relates to a process comprisingoxidizing a compound selected from the group consisting ofN-substituted-1-deoxy-1-hexosamines based on mannose, allose andaltrose, N-substituted-1-deoxy-1-pentosamines,N-substituted-1-deoxy-1-tetrosamines, and salts thereof, with anoxidizing microbe selected from the group consisting of bacteria of thefamily Acetobacteraceae, bacteria of the genus Corynebacterium, and cellfragments or cell free extracts thereof, producing a correspondingcompound selected from the group consisting ofN-substituted-amino-6-deoxy-2-ketohexuloses, based on mannose, alloseand altrose, N-substituted-amino-5-deoxy-2-ketopentuloses,4-(N-substituted)-amino-1,3-dihydroxy-2-butanones, and salts thereof,and then reducing the oxidized compound to produce the correspondingcompound selected from the group consisting of N-substituted polyhydroxypiperidines based on N-substituted mannosamines, allosamines andaltrosamines, N-substituted polyhydroxy pyrrolidines, N-substitutedpolyhydroxy azetidines, and salts thereof.

A third embodiment of the invention relates to a process comprisingamination of a sugar selected from the group consisting of mannose,allose, altrose, ribose, arabinose and erythrose to produce thecorresponding amino compound selected from the group consisting ofN-substituted-1-deoxy-1-hexosamines based on mannose, allose andaltrose, N-substituted-1-deoxy-1-pentosamines,N-substituted-1-deoxy-1-tetrosamines, and salts thereof, oxidizing theamino compound with a microbe selected from the group consisting ofbacteria of the family Acetobacteraceae, bacteria of the genusCorynebacterium, and cell fragments or cell free extracts thereof,producing a corresponding compound selected from the group consisting ofN-substituted-amino-6-deoxy-2-ketohexuloses based on mannose, allose andaltrose, N-substituted-amino-5-deoxy-2-ketopentuloses,4-(N-substituted)-amino-1,3-dihydroxy-2-butanones, and salts thereof,and then reducing said oxidized compound to produce the correspondingcompound selected from the group consisting of N-substituted polyhydroxypiperidines based on N-substituted mannosamines, allosamines andaltrosamines, N-substituted polyhydroxy pyrrolidines, N-substitutedpolyhydroxy azetidines, and salts thereof.

A fourth embodiment of the invention relates to a 1-pot process whichcomprises the steps of (a) mixing a solvent and an amine, (b) adjustingthe pH to about 8.0 to about 12.0, (c) adding a sugar selected from thegroup consisting of mannose, allose, altrose, ribose, arabinose anderythrose in about a 1:1 ratio with the amine, (d) adding a catalyst,(e) reducing at a pressure of about 1 to about 100 atm and a temperatureof about 25° C. to about 100° C., (f) removing the catalyst, (g)adjusting the pH to about 1 to about 7, and (h) removing the solvent toobtain the corresponding salt selected from the group consisting ofN-substituted-1-deoxy-1-hexosamines based on mannose, allose andaltrose, N-substituted-1-deoxy-1-pentosamines, andN-substituted-1-deoxy-1-tetrosamines. The residue containing thecorresponding salt is diluted with water and is ready for use in thenext step of microbial oxidation without purification.

This can be demonstrated by the following examples.

When D-mannose is used as the starting sugar: ##STR1##

When D-ribose is used as the starting sugar: ##STR2##

When D-erythrose is used as the starting sugar: ##STR3##

The exact form of the structure of formulas II, V and VIII is dictatedby the environment in which the oxidized compound is present (See H.Paulsen et al., Chem. Ber.100:802 (1967)). The use of thefructofuranose, erythropentulose and butanone nomenclature is not meantto imply that the compound cannot or does not exist in another of itsequivalent forms.

The products of the microbial oxidation of the invention are useful asintermediates for producing the N-substituted polyhydroxy piperidinesbased on N-substituted derivatives of mannosamine, allosamine andaltrosamine, polyhydroxy pyrrolidines and polyhydroxy azetidines of theinvention which are believed to have utility as antiviral agents,antidiuretics, antidiabetics, animal feed additives andantihyperglycemics.

The substituent on the nitrogen in any of the compounds of the inventionis selected from the group consisting of hydrogen, phenyl, C₁ -C₁₀alkyl, C₁ -C₁₀ alkyl substituted with aromatic, amide or carboxyradicals, and C₂ -C₁₀ alkyl substituted with hydroxy radicals.

Straight chain or branched chain alkyls are suitable to practice theprocess of the invention, with C₁ -C₅ alkyl groups preferred. Examplesof suitable alkyl radicals are methyl, ethyl, n-propyl, 1-methylethyl,n-butyl, 1-methylpropyl, 1,1-dimethylethyl, n-pentyl, 3-methylbutyl,1-methylbutyl, 2-methylbutyl, n-hexyl, n-heptyl, n-octyl, n-nonyl andn-decyl. Suitable hydroxy substituted alkyl radicals are 2-hydroxyethyl,3-hydroxypropyl, 4-hydroxybutyl, 5-hydroxypentyl, 6-hydroxyhexyl,7-hydroxyheptyl, 8-hydroxyoctyl, 9-hydroxynonyl, and 10-hydroxydecyl.Suitable carboxy substituted alkyl radicals are carboxymethyl,2-carboxyethyl, 3-carboxypropyl, 4-carboxybutyl, 5-carboxypentyl,6-carboxyhexyl, 7-carboxyheptyl, 8-carboxyoctyl, 9-carboxynonyl and10-carboxydecyl. Suitable aromatic substituted alkyl radicals arephenylmethyl (benzyl), 2-phenylethyl, 3-phenylpropyl, 4-phenylbutyl,5-phenylpentyl, 6-phenylhexyl, 7-phenylheptyl, 8-phenyloctyl,9-phenylnonyl, 10-phenyldecyl and 2-naphthylmethyl. Phenyl alone is alsoan acceptable radical.

Examples of N-substituted-1-deoxy-1-hexosamines based on mannose, alloseand altrose, N-substituted-1-deoxy-1-pentosamines, andN-substituted-1-deoxy-1-tetrosamines of the invention include, but arenot limited to:

N-benzyl-1-deoxy-1-mannosamine

N-(2-naphthylmethyl)-1-deoxy-1-mannosamine

N-butyl-1-deoxy-1-mannosamine

N-benzyl-1-deoxy-1-altrosamine

N-(2-naphthylmethyl)-1-deoxy-1-altrosamine

N-butyl-1-deoxy-1-altrosamine

N-butyl-1-deoxy-1-allosamine

N-benzyl-1-deoxy-1-allosamine

N-(2-naphthylmethyl)-1-deoxy-1-allosamine

N-benzyl-1-deoxy-1-ribosamine

N-(2-naphthylmethyl)-1-deoxy-1-ribosamine

N-butyl-1-deoxy-1-ribosamine

N-benzyl-1-deoxy-1-arabinosamine

N-(2-naphthylmethyl)-1-deoxy-1-arabinosamine

N-butyl-1-deoxy-1-arabinosamine

N-benzyl-1-deoxy-1-erythrosamine

N-(2-naphthylmethyl)-1-deoxy-1-erythrosamine

N-butyl-1-deoxy-1-erythrosamine

Examples of N-substituted-amino-6-deoxy-2-ketohexuloses based onmannose, allose and altrose,N-substituted-amino-5-deoxy-2-ketopentuloses, and4-(N-substituted)-amino-1,3-dihydroxy-2-butanones, produced by themicrobial oxidation process of the invention include but are not limitedto:

6-butylamino-6-deoxy-D-fructofuranose

6-benzylamino-6-deoxy-D-fructofuranose

6-(2-naphthylmethylamino)-6-deoxy-D-fructofuranose

6-butylamino-6-deoxy-D-tagatofuranose

6-benzylamino-6-deoxy-D-tagatofuranose

6-(2-naphthylmethylamino)-6-deoxy-D-tagatofuranose

6-butylamino-6-deoxy-L-psicofuranose

6-benzylamino-6-deoxy-L-psicofuranose

6-(2-naphthylmethylamino)-6-deoxy-L-psicofuranose

5-butylamino-5-deoxy-L-erythro-2-pentulose

5-benzylamino-5-deoxy-L-erythro-2-pentulose

5-(2-naphthylmethylamino)-5-deoxy-L-erythro-2-pentulose

5-butylamino-5-deoxy-D-threo-2-pentulose

5-benzylamino-5-deoxy-D-threo-2-pentulose

5-(2-naphthylmethylamino)-5-deoxy-D-threo-2-pentulose

4-butylamino-(S)-1,3-dihydroxy-2-butanone

4-benzylamino-(S)-1,3-dihydroxy-2-butanone

4-(2-naphthylmethyl)-(S)-1,3-dihydroxy-2-butanone

Examples of N-substituted polyhydroxy piperidines based on N-substitutedmannosamines, allosamines and altrosamines, N-substituted polyhydroxypyrrolidines, and N-substituted polyhydroxy azetidines that can beproduced by hydrogenating the oxidized compounds produced by themicrobial oxidation process of the invention include but are not limitedto:

1-benzyl-2-hydroxymethyl- 3R-(3α, 4β, 5β)!-3,4,5-piperidinetriol

1-butyl-2-hydroxymethyl- 3R-(3α, 4β, 5β)!-3,4,5-piperidinetriol

1-(2-naphthylmethyl)-2-hydroxymethyl- 3R-(3α, 4β,5β)!-3,4,5-piperidinetriol

1-benzyl-2-hydroxymethyl- 3R-(3α, 4α, 5β)!-3,4,5-piperidinetriol

1-butyl-2-hydroxymethyl- 3R-(3α, 4α, 5β)!-3,4,5-piperidinetriol

1-(2-naphthylmethyl)-2-hydroxymethyl- 3R-(3α, 4α,5β)!-3,4,5-piperidinetriol

1-benzyl-2-hydroxymethyl- 3R-(3α, 4α, 5α)!-3,4,5-piperidinetriol

1-butyl-2-hydroxymethyl- 3R-(3α, 4α, 5α)!-3,4,5-piperidinetriol

1-(2-naphthylmethyl)-2-hydroxymethyl- 3R-(3α, 4α,5α)!-3,4,5-piperidinetriol

1-benzyl-2-hydroxymethyl-(3R-cis)-3,4,-pyrrolidinediol

1-butyl-2-hydroxymethyl-(3R-cis)-3,4,-pyrrolidinediol

1-(2-naphthylmethyl)-2-hydroxymethyl-(3R-cis)-3,4,-pyrrolidinediol

1-benzyl-2-hydroxymethyl-(3R-trans)-3,4,-pyrrolidinediol

1-butyl-2-hydroxymethyl-(3R-trans)-3,4,-pyrrolidinediol

1-(2-naphthylmethyl)-2-hydroxymethyl-(3R-trans)-3,4,-pyrrolidinediol

1-benzyl-2-hydroxymethyl-3(S)-hydroxyazetidine

1-butyl-2-hydroxymethyl-3(S)-hydroxyazetidine

1-(2-naphthylmethyl)-2-hydroxymethyl-3(S)-hydroxyazetidine

The N-substituted amino compounds, i.e. theN-substituted-1-deoxy-1-hexosamines based on mannose, allose andaltrose, N-substituted-1-deoxy-1-pentosamines,N-substituted-1-deoxy-1-tetrosamines, and salts thereof, of theinvention can be obtained by known means, for example, by amination ofthe respective sugars. The reductive alkylation of sugars with amines isreported in the literature as a method for preparingN-substituted-1-amino-1-deoxy sugars (see F. Kagan et al., J. Amer.Chem. Soc., 79, 3541 (1957), A. Mohammad et al., J. Am. Chem. Soc., 66,969 (1947), P. N. Rylander, Hydrogenation Methods (Academic Press,(1985) pp. 82-93) and G. Mitts et al., J. Am. Chem. Soc., 66:483(1944)). In general these preparations involve reacting a sugar and anamine, in varying ratios, in a suitable solvent such as aqueous methanolor ethanol with an appropriate catalyst such as Raney nickel orpalladium on carbon. A catalytic amount of hydrochloric acid issometimes added. The resulting mixture is hydrogenated under 40-1300psig of hydrogen pressure at 23°-100° C. for 7-30 hours. The resultingN-substituted amino compound is then isolated.

In a preferred process for preparing N-substituted amino compounds, aParr shaker bottle, or the like, is charged with a solvent and amine.Suitable solvents include water, alcohols (such as methanol and ethanol)or aqueous alcohols. Preferably the solvent is ethanol. Suitable aminesinclude but are not limited to methyl amine, phenyl amine, ethyl amine,propyl amine, 1-methylethyl amine, n-butyl amine, methylpropyl amine,1,1-dimethylethyl amine, n-pentyl amine, 3-methylbutyl amine,1-methylbutyl amine, 2-methylbutyl amine, n-hexylamine, n-heptyl amine,n-octyl amine, n-nonyl amine, n-decyl amine, 2-hydroxyethyl amine,4-carboxybutyl amine, benzyl amine, 5-phenylpentyl amine, 6-phenylhexylamine, 7-phenylheptyl amine, 8-phenyloctyl amine, 9-phenylnonyl amine,10-phenyldecyl amine, 2-(aminomethyl)naphthalene, and4-(aminomethyl)pyridine. Preferred amines include ethyl amine, n-butylamine, n-octyl amine, 2-hydroxyethyl amine, benzyl amine, phenyl amine,2-(aminomethyl)naphthalene and 4-carboxybutyl amine. The ratio of sugarto amine is about 1:1, which allows the product to be used withoutisolation or removal of excess reagents. The mixture is stirred andcooled while acid is slowly added until a pH in the range of about 8.0to about 12.0 is obtained, preferably about 9 to about 10.5. Suitableacids include hydrochloric acid, sulfuric acid, nitric acid, aceticacid, ascorbic acid, succinic acid, citric acid, maleic acid, oxalicacid, and phosphoric acid, preferably hydrochloric acid. To the Parrshaker bottle is added the sugar followed by palladium-on-carbon (Pd/C)catalyst (50% water-wet). A palladium catalyst loading of about 1% toabout 50% by weight sugar is used, preferably about 10% to about 30%.Catalysts, including but not limited to, Raney nickel, platinum,palladium, rhodium and rhenium, preferably palladium and Raney nickel,can be used. The mixture is agitated and hydrogenated at a pressure ofabout 1 to about 100 atm, preferably about 3 to about 6 atm of hydrogenand a temperature of about 25° C. to about 100° C., preferably about 40°C. to about 80° C., until the reaction is complete (as indicated byhydrogen uptake). The hydrogen is vented and the palladium-on-carbonremoved by filtration (preferably through a layer of powderedcellulose). The catalyst is washed with solvent such as an alcohol,preferably ethanol, followed by washing with water. The washes arecombined with the filtrate to give a solution containing a mixture ofN-substituted amino compound and its corresponding salt. The solution iscooled to crystallize the amino sugar which is isolated. Alternatively,the mixture is stirred and cooled while hydrochloric acid is slowlyadded to a final pH of about 1 to about 7, preferably about 4 to about6. The ethanol is removed by distillation under reduced pressure. Theresidue contains the salt of the N-substituted amino compound. Theresidue is diluted with water and ready to use in the next step ofmicrobial oxidation without purification. Thus, the process produces theN-substituted amine compound salts from the respective sugars withoutisolation or removal of excess reagents. The elimination of isolationand excess reagent removal steps allows for the direct use ofN-substituted amino compounds in the microbial oxidation, whichoxidation results in the 6-deoxy-6-(N-substituted)-amino-2-hexulosesbased on mannose, allose and altrose,5-deoxy-5-(N-substituted)-amino-2-pentuloses and4-(N-substituted)-amino-1,3-dihydroxy-2-butanones which in turn can bedirectly hydrogenated to N-substituted polyhydroxy piperidines based onN-substituted mannosamines, allosamines and altrosamines, N-substitutedpolyhydroxy pyrrolidines, and N-substituted polyhydroxy azetidines (i.e.one pot process).

An additional advantage of the N-substituted amino compound salts is theelimination of odor associated with residual amines. Typically theamines are extremely odoriferous, requiring the use of respirators whenhandling. On the other hand, the amino compound salts are relativelyodor free, which enables handling without special precautions such asrespirators.

As indicated by Material Safety Data Sheets from suppliers ofn-butylamine (Fisher Scientific, Fair Lawn, N.J., for example), then-butylamine compound is toxic and a severe eye, skin and mucousmembrane irritant. Exposure to as little as 5-10 ppm of n-butylamineproduces nose and throat irritation. Exposure to concentrations of 10-25ppm are intolerable for more than a few minutes. Thus, the salt forms ofthe N-substituted amino compounds, which forms do not have the odor andirritation characteristics of the non-salt forms, are advantageous.

To begin the microbial oxidation of an N-substituted amino compound,microorganisms are added to a reaction mixture which comprises anN-substituted amino compound or salts thereof. Alternatively,N-substituted amino compound or a salt thereof is added to cultures ofmicroorganisms that will carry out the oxidation step. Preferably a saltof N-substituted amino compound is added. Suitable salts ofN-substituted amino compounds include but are not limited to salts ofchloride, sulfate, nitrate, acetate, ascorbate, succinate, citrate,maleate, oxalate, or phosphate. Preferably the hydrochloride salt isused. Although the use of a salt is preferred, a salt can be made insitu by the addition of an N-substituted amino compound and suitableacids to lower the pH and create an N-substituted amino compound salt.During incubation of the reaction mixture containing microorganisms, thereaction is monitored with a reverse phase or ion exchange highperformance liquid chromatography (HPLC) assay to observe conversion ofN-substituted amino compound to the respectiveN-substituted-amino-6-deoxy-2-ketohexuloses based on mannose, allose andaltrose, N-substituted-amino-5-deoxy-2-ketopentuloses, and4-(N-substituted)-amino-1,3-dihydroxy-2-butanones. Thin layerchromatography (TLC) and gas chromatography (GC) can also be used tomonitor the conversion.

Microorganisms which are suitable for carrying out the oxidation (ormicroorganisms from which active cell fragments or cell free extractsfor carrying out the oxidation are obtained) can be Procaryotae(bacteria), or Eucaryotae, for example fungi, which in each case canbelong to diverse taxonomic groups. Suitable microorganisms are found bygrowing a relatively large number of microorganisms in an appropriatenutrient medium which contains N-substituted amino compounds andexamining their ability to produce the oxidized N-substituted aminocompounds. The ability of a microorganism to catalyze the oxidationreaction according to the invention can be measured by a variety ofmeans, including assaying with high performance liquid chromatography(HPLC). Microorganisms for use in the process of the invention arereadily available from a variety of sources including but not limited tothe American Type Culture Collection (ATCC), Rockville, Md.; theAgricultural Research Culture Collection (NRRL), Peoria, Ill.; DeutscheSammlung Von Mikroorganismen (DSM), Federal Republic of Germany; and theFermentation Research Institute (FRI), Japan. Alternatively, arecombinant microorganism can be prepared by isolating or synthesizingthe appropriate gene for the oxidizing enzyme and inserting this geneinto another microorganism using standard literature techniques such asis disclosed in Molecular Cloning, A Laboratory Manual, 2nd Edition, J.Sambrook, E. F. Fritsch, T. Maniatis, eds, Vol. 1,2, and 3, Cold SpringHarbor Laboratory Press (1989).

Examples of suitable microorganisms which are readily available from theabove-identified culture collections are bacteria from the orderPseudomonadales and cell fragments or cell free extracts therefrom,bacteria from the family Acetobacteraceae and cell fragments or cellfree extracts therefrom, bacteria from the family Coryneform and cellfragments or cell free extracts therefrom, and fungi from the genusMetschnikowia. Within the Pseudomonadales order, preference is forrepresentatives of the family Acetobacteraceae. Within theAcetobacteraceae family, bacteria of the genus Gluconobacter (formerlycalled Acetobacter) are preferred. Bacteria from the group of Coryneformbacteria, in particular those of the genus Corynebacterium (also knownas Curtobacterium), are also suitable. Finally, the oxidation can becarried out with fungi (for example, with yeasts) in particular withthose of the family Spermophthoraceae, such as the genus Metschnikowia.In addition, fungi from the genera Agarius and Cephalosporium, andyeasts from the genera Candida and Saccharomyces can be used in theinvention.

Examples of suitable Corynebacterium are Corynebacteriumacetoacidophilum, Corynebacterium acetoglutamicum, Corynebacteriumacnes, Corynebacterium alkanolyticum, Corynebacterium alkanum,Corynebacterium betae (also known as curtobacterium betae),Corynebacterium bovis, Corynebacterium callunae, Corynebacteriumcystitidis, Corynebacterium dioxydans, Corynebacterium equi,Corynebacterium flavescens, Corynebacterium glutamicum, Corynebacteriumherculis, Corynebacterium hoagii, Corynebacterium hydrocarbooxydans,Corynebacterium ilicis, Corynebacterium lilium, Corynebacteriumliquefaciens, Corynebacterium matruchotii, Corynebacterium melassecola,Corynebacterium mycetoides, Corynebacterium nephridii, Corynebacteriumnitrilophilus, Corynebacterium oortii, Corynebacterium petrophilum,Corynebacterium pilosum, Corynebacterium pyogenes, Corynebacteriumrathayi, Corynebacterium renale, Corynebacterium simplex,Corynebacterium striatum, Corynebacterium tritici, Corynebacteriumuratoxidans, Corynebacterium vitarumen, and Corynebacterium xerosis.Suitable Gluconobacterium for use in the process of the inventioninclude Gluconobacter oxydans subsp. industrius, Gluconobacter oxydanssubsp. melanogenes, Gluconobacter oxydans subsp. sphaericus, andGluconobacter oxydans subsp. suboxydans. Suitable Acetobacterium for usein the process of the invention include Acetobacter aceti, Acetobacterhansenii, Acetobacter liquefaciens (formerly called Gluconobacterliquefaciens), Acetobacter methanolicus, Acetobacter pasteurianus andAcetobacter sp. . Metschnikowia (formerly called Candida) preferred foruse in the process of the invention include Metschnikowia pulcherrimiaand yeasts such as Candida utilis and Saccharomyces cerevisiae.

General growth conditions for culturing the particular organisms areobtained from depositories and from texts known in the art such asBergey's Manual of Systematic Bacteriology, Vol.1, Williams and Wilkins,Baltimore/London (1984), N. R. Krieg, ed.

The nutrient medium for the growth of any oxidizing microorganism shouldcontain sources of assimilable carbon and nitrogen, as well as mineralsalts. Suitable sources of assimilable carbon and nitrogen include, butare not limited to, complex mixtures, such as those constituted bybiological products of diverse origin, for example soy bean flour,cotton seed flour, lentil flour, pea flour, soluble and insolublevegetable proteins, corn steep liquor, yeast extract, peptones and meatextracts. Additional sources of nitrogen are ammonium salts andnitrates, such as ammonium chloride, ammonium sulphate, sodium nitrateand potassium nitrate. Generally, the nutrient medium should include,but is not limited to, the following ions: Mg⁺⁺, Na⁺, K⁺, Ca⁺⁺, NH₄ ⁺.Cl⁻, SO₄ ⁻⁻, PO₄ ⁻⁻⁻ and NO₃ ⁻ and also ions of the trace elements suchas Cu, Fe, Mn, Mo, Zn, Co and Ni . The preferred source of these ionsare mineral salts.

If these salts and trace elements are not present in sufficient amountsin the complex constituents of the nutrient medium or in the water usedit is appropriate to supplement the nutrient medium accordingly.

The microorganism employed in the process of the invention can be in theform of fermentation broths, whole washed cells, concentrated cellsuspensions, cell fragments or cell free extracts, and immobilizedcells. Preferably concentrated cell suspensions, cell fragments or cellfree extracts, and whole washed cells are used with the process of theinvention (S. A. White and G. W. Claus (1982), J. Bacteriology,150:934-943 and S. Berezenko and R. J.Sturgeon (1991), CarbohydrateResearch, 216: 505-509).

Concentrated washed cell suspensions can be prepared as follows: Themicroorganisms are cultured in a suitable nutrient solution, harvested(for example by centrifuging) and suspended in a smaller volume (in saltor buffer solutions, such as physiological sodium chloride solution oraqueous solutions of potassium phosphate, sodium acetate, sodiummaleate, magnesium sulfate, or simply in tap water, distilled water ornutrient solutions). N-substituted amino compound or a salt thereof isthen added to a cell suspension of this type and the oxidation reactionaccording to the invention is carried out under the conditionsdescribed.

The conditions for oxidation of N-substituted amino compound in growingmicroorganism cultures or cell fragments or cell free extracts aresimilar to those for carrying out the process according to the inventionwith concentrated cell suspensions. In particular the temperature rangeis from about 0° C. to about 45° C. and the pH range is from about 2 toabout 10. There are no special nutrients necessary in the process of theinvention. More importantly, washed or immobilized cells, cell fragmentsor cell free extracts can simply be added to a solution of N-substitutedamino compound or salts thereof, without any nutrient medium present.

The process of the invention can also be carried out with cell fragmentsor cell free extracts prepared from bacteria. The cell free extracts canbe crude extracts, such as obtained by conventional digestion ofmicroorganism cells. Methods to break up cells include, but are notlimited to, mechanical disruption, physical disruption, chemicaldisruption, and enzymatic disruption. Such means to break up cellsinclude ultrasonic treatments, passages through French pressure cells,grindings with quartz sand, autolysis, heating, osmotic shock, alkalitreatment, detergents, or repeated freezing and thawing.

If the process according to the invention is to be carried out withpartially purified cell fragments or cell free extract preparations, themethods of protein chemistry, such as ultracentrifuging, precipitationreactions, ion exchange chromatography or adsorption chromatography, gelfiltration or electrophoretic methods, can be employed to obtain suchpreparations. In order to carry out the reaction according to theinvention with fractionated cell free extracts, it may be necessary toadd to the system additional reactants such as, physiological orsynthetic electron acceptors, like NAD⁺, NADP⁺, methylene blue,dichlorophenolindophenol, tetrazolium salts and the like. When thesereactants are used, they can be employed either in equimolar amounts(concentrations which correspond to that of the N-substituted aminocompound employed) or in catalytic amounts (concentrations which aremarkedly below the chosen concentration of N-substituted aminocompound). If, when using catalytic amounts, it is to be ensured thatthe process according to the invention is carried out approximatelyquantitatively, a system which continuously regenerates the reactantwhich is present only in a catalytic amount must also be added to thereaction mixture. This system can be, for example, an enzyme whichensures reoxidation (in the presence of oxygen or other oxidizingagents) of an electron acceptor which is reduced in the course of thereaction according to the invention.

If nutrient media is used with intact microorganisms in a growingculture, nutrient media can be solid, semi-solid or liquid.Aqueous-liquid nutrient media are preferably employed when media isused. Suitable media and suitable conditions for cultivation includeknown media and known conditions to which N-substituted amino compoundor salts thereof can be added.

The N-substituted amino compound or salts thereof to be oxidized in theprocess according to the invention can be added to the base nutrientmedium either on its own or as a mixture with one or more oxidizablecompounds. Additional oxidizable compounds which can be used includepolyols, such as sorbitol or glycerol.

If one or more oxidizable compounds are added to the nutrient solution,the N-substituted amino compound or salts thereof to be oxidized can beadded either prior to inoculation or at any desired subsequent time(between the early log phase and the late stationary growth phase). Insuch a case the oxidizing organism is pre-cultured with the oxidizablecompounds. The inoculation of the nutrient media is effected by avariety of methods including slanted tube cultures and flask cultures.

Contamination of the reaction solution should be avoided. To avoidcontamination, sterilization of the nutrient media, sterilization of thereaction vessels and sterilization of the air required for aerationshould be undertaken. It is possible to use, for example, steamsterilization or dry sterilization for sterilization of the reactionvessels. The air and the nutrient media can likewise be sterilized bysteam or by filtration. Heat sterilization of the reaction solutioncontaining the substrates (N-substituted amino compound) is alsopossible.

The process of the invention can be carried out under aerobic conditionsusing shake flasks or aerated and agitated tanks. Preferably, theprocess is carried out by the aerobic submersion procedure in tanks, forexample in conventional fermentors. It is possible to carry out theprocess continuously or with batch or fed batch modes, preferably thebatch mode.

It is advantageous to ensure that the microorganisms are adequatelybrought into contact with oxygen and the N-substituted amino compounds.This can be effected by several methods including shaking, stirring andaerating.

If foam occurs in an undesired amount during the process, chemical foamcontrol agents, such as liquid fats and oils, oil-in-water emulsions,paraffins, higher alcohols (such as octadecanol), silicone oils,polyoxyethylene compounds and polyoxypropylene compounds, can be added.Foam can also be suppressed or eliminated with the aid of mechanicaldevices.

The time-dependent formation of the oxidized N-substituted aminocompounds, i.e., N-substituted-amino-6-deoxy-2-ketohexuloses based onmannose, allose and altrose,N-substituted-amino-5-deoxy-2-ketopentuloses, and4-(N-substituted)-amino-1,3-dihydroxy-2-butanones, in the culture mediumcan be followed either by thin layer chromatography or HPLC. Preferablythe time-dependent formation of the oxidized N-substituted aminocompounds is measured by HPLC.

The oxidized N-substituted amino compound obtained in accordance withthe process of the invention is isolated from the reaction solution asfollows: The cell mass is filtered off or centrifuged off and thesupernatant liquor is passed through a column containing acid ionexchanger and rinsed with an alcohol or water. Elution is then carriedout with a base and the eluate concentrated. After adding acetone or thelike, the oxidized N-substituted amino compound crystallizes out. If itis intended to carry out further processing of oxidized N-substitutedamino compound to N-substituted polyhydroxy piperidines based onN-substituted mannosamines, allosamines and altrosamines, N-substitutedpolyhydroxy pyrrolidines, and N-substituted polyhydroxy azetidines,isolation and/or recovery is not necessary. For producing N-substitutedpolyhydroxy piperidines based on N-substituted mannosamines, allosaminesand altrosamines, N-substituted polyhydroxy pyrrolidines, andN-substituted polyhydroxy azetidines from oxidized N-substituted aminocompound, the clear solution, after removal of the cell mass, isreduced, preferably in the presence of a catalyst.

This aspect of the invention (no isolation and/or recovery necessary) isparticularly advantageous because the process proceeds directly from thesupernatant liquor resulting from the removal of cell mass of themicrobial oxidation reaction solution. Likewise, it is especiallyadvantageous because, unlike prior art processes, no amino protectinggroup has to be removed. The process of the invention eliminates theneed to make and isolate protecting group intermediates and avoidsremoval of the protecting group to obtain the desired compound. Theelimination of these steps results in a more efficient process withgreater conversions and overall yields, less equipment and shorter cycletimes. The oxidized N-substituted amino compounds also exhibit highersolubility, thus higher concentrations are obtainable, which results inhigh productivity and higher rates. In addition, the oxidizedN-substituted amino compounds have great stability which impedesdegradation and resulting byproducts.

Several known means are available for reduction (see for example P. N.Rylander, Hydrogenation Methods (Academic Press, (1985) pp 82-93 andOrganic Chemistry, 3rd edition, Eds James B. Hendrickson, Donald J.Cram, George S. Hammond (McGraw-Hill, Chapter 18, 1970)). These meansinclude metal hydride reduction, catalytic hydrogenation, dissolvingmetal reduction and electrochemical reduction. In general, to reduce theoxidized N-substituted amino compounds to N-substituted polyhydroxypiperidines based on N-substituted mannosamines, allosamines andaltrosamines, N-substituted polyhydroxy pyrrolidines, and N-substitutedpolyhydroxy azetidines, the oxidized N-substituted amino compound ischarged to a flask followed by addition of decolorizing carbon. Thestirred mixture is then filtered to remove the carbon. The filtrate isadded to a hydrogenation apparatus, such as a Parr Laboratory Reactor,containing a hydrogenation catalyst. Catalyst loading from about 1-100%by weight of the oxidized N-substituted amino compound using Group VIIIB metals are used. Preferably about 40-60% is used. Such catalystsinclude but are not limited to palladium, platinum, nickel and rhodium.Supports for the catalysts may include but are not limited to alumina,barium sulfate, calcium carbonate, carbon, silica and kieselguhr.Typically, the support would contain a 1-20% metal loading, preferably a4-10% loading. A palladium catalyst is preferred. The mixture ishydrogenated for about 5 hours. Hydrogen pressure from about 1 to about100 atm can be used; preferably a range from about 1 to about 5 atm isused. The catalyst is then removed and acid ion-exchange resin added tothe filtrate to adsorb the N-substituted polyhydroxy piperidines basedon N-substituted mannosamines, allosamines and altrosamines,N-substituted polyhydroxy pyrrolidines, and N-substituted polyhydroxyazetidines. The N-substituted polyhydroxy piperidines based onN-substituted mannosamines, allosamines and altrosamines, N-substitutedpolyhydroxy pyrrolidines, and N-substituted polyhydroxy azetidines arereleased from the resin and isolated.

When the substituent on the nitrogen of the oxidized N-substituted aminocompounds is methyl substituted with aromatic, the oxidizedN-substituted amino compounds can be reduced directly to thecorresponding N-substituted polyhydroxy piperidines based onN-substituted mannosamines, allosamines and altrosamines, N-substitutedpolyhydroxy pyrrolidones, and N-substituted polyhydroxy azetidineswherein the substituent on the nitrogen is hydrogen by using catalytichydrogenation such as with a palladium on carbon catalyst. Thecorresponding N-substituted polyhydroxy piperidines based onN-substituted mannosamines, allosamines and altrosamines, N-substitutedpolyhydroxy pyrrolidones, and N-substituted polyhydroxy azetidineswherein the substituent on the nitrogen is methyl substituted witharomatic can be prepared by using metal hydrides as the reducing agent.

The following examples illustrate the specific embodiments of theinvention described herein. As would be apparent to skilled artisans,various changes and modifications are possible and are contemplatedwithin the scope of the invention described.

EXAMPLES Example 1 Preparation of Cell Paste of Microorganisms

A Gluconobacter oxydans cell paste is prepared by inoculating a seriesof 10 liter fermentors, each containing eight liters of media with 60gm. D-sorbitol/liter, 24 gm. yeast extract/liter, 48 gm. potassiumphosphate dibasic/liter and 0.3 ml. antifoam/liter (Ucon LB 625) withthe microorganism G. oxydans (DSM2003). The fermentors are agitated andaerated while controlling temperature (30° C.) and pH (5.5 to 6.5)during the cell growth period. The fermentations are terminated afterabout 27 hours when optical density measurements indicate the log growthphase has been completed. The broths are then cooled, centrifuged, andthe cells resuspended in water (or 0.02M MgSO4) and centrifuged toproduce washed cell paste. These cell pastes are subdivided intoaliquots and stored at or below 10° C. until thawed for addition to areaction solution.

2.90 Grams of N-benzyl-1-deoxy-1-mannosamine were dissolved in 50 mLwater and the pH adjusted to 5.0 with concentrated HCl. This solutionwas filtered through a 0.2μ filter and placed in a sterile 500 mL shakeflask. To this were added freshly thawed Gluconobacter oxydans cells togive approximately 47 mg/mL (wet cell weight). The shake flaskcontaining the suspension was rotated at 120 rpm, room temperature for22 hours. At this point the suspension was clarified by centrifugationand filtered through a 0.2μ filter. 13.5 mL of this solution was chilledin ice, the pH adjusted to over 11 with 1.5 mL 2.5N NaOH and reducedwith 233 mg NaBH₄ at 0° C. for two hours. After standing overnight underrefrigeration, the solution was acidified and freeze-dried. Afteracetylation of a sample with 1:1 triethylamine-acetic anhydride, GC-massspec indicated the presence of a peak at m/e 422, corresponding to M+Hfor 1-benzyl-2-hydroxymethyl- 3R-(3α, 4β, 5β)!-3,4,5-piperidinetrioltetraacetate.

Example 2

25 mL of the bioconversion solution from Example 1 was reduced withhydrogen and Pd/C at 63 psig, room temperature for 2.5 hours. Afterlyophilization and acetylation of a sample, GC-mass spec indicated thepresence of a peak at m/e 374, corresponding to M+H for 2-hydroxymethyl-3R-(3α, 4β, 5β)!-3,4,5-piperidinetriol pentaacetate.

Example 3

3.49 Grams of N-(2-naphthylmethyl)-1-deoxy-1-mannosamine were dissolvedin 50 mL water and the pH adjusted to 5.0 with concentrated HCl. Thissolution was filtered through a 0.2μ filter and placed in a sterile 500mL shake flask. To this were added freshly thawed Gluconobacter oxydanscells to give approximately 54 mg/mL (wet cell weight). The shake flaskcontaining the suspension was rotated at 120 rpm, room temperature for22 hours. At this point the suspension was clarified by centrifugationand filtered through a 0.2μ filter. 12.5 mL of this solution was chilledin ice, the pH adjusted to 8.1 with 1.2 mL 2.5N NaOH, 25 mL of chilledmethanol were added and the sample reduced with 318 mg NaBH₄ at 0° C.for two hours. After standing overnight under refrigeration, thesolution was acidified and freeze-dried. Acetylation of a sample of thedried product gave m/e 472 (M+H) by GC-mass spec, corresponding to1-(2-naphthylmethyl)-2-hydroxymethyl- 3R-(3α, 4β,5β)!-3,4,5-piperidinetriol tetraacetate.

Example 4

25 mL of the bioconversion solution from Example 3 was reduced withhydrogen using a Pd/C catalyst at 63 psig, room temperature for 3.5hours. After lyophilization and acetylation of a sample, GC-mass specindicated the presence of a peak at m/e 374, corresponding to M+H forthe pentaacetate of 2-hydroxymethyl- 3R-(3α, 4β,5β)!-3,4,5-piperidinetriol.

Example 5

1 Gram of N-butyl-1-deoxy-1-mannosamine was dissolved in 40 mL water andthe pH adjusted to 5.0 with HCl, and the volume adjusted to 50 mL. Thissolution was filtered through a 0.2μ filter and placed in a sterile 500mL shake flask. To this were added freshly thawed Gluconobacter oxydanscells to give approximately 50 mg/mL (wet cell weight). The shake flaskcontaining the suspension was rotated at 120 rpm, room temperature for48 hours. At this point the suspension was clarified by centrifugationand filtered through a 0.2μ filter and freeze-dried. HPLC assay after 24hours indicated over 95% conversion to6-butylamino-6-deoxy-D-fructofuranose.

Example 6

1.5 Grams of N-butyl-1-deoxy-1-arabinosamine hydrochloride was dissolvedin 45 mL water and (the pH was 5.35) the solution filtered through a0.45μ filter and placed in a sterile 500 mL shake flask. To this wereadded freshly thawed Gluconobacter oxydans cells to give approximately40 mg/mL (wet cell weight). The shake flask containing the suspensionwas rotated at 120 rpm, room temperature for 48 hours. After 48 hours,the suspension was clarified by centrifugation. After treatment withcharcoal, catalytic hydrogenation (4% Pd/C) at 50 psig and acetylation,GC-mass spec indicated the presence of a peak with m/e at 316,consistent with1-(n-butyl)-2-hydroxymethyl-(3R-trans)-3,4-pyrrolidinediol triacetate.

Example 7

1 Gram of N-benzyl-1-deoxy-1-arabinosamine was dissolved in 45 mL waterand the pH adjusted to 5.1 with HCl. This solution was filtered througha 0.45μ filter and placed in a sterile 500 mL shake flask. To this wereadded freshly thawed Gluconobacter oxydans cells to give approximately40 mg/mL (wet cell weight). The shake flask containing the suspensionwas rotated at 120 rpm, room temperature for 48 hours, at which time thesuspension was clarified by centrifugation. The supernatant was treatedwith charcoal and catalytically hydrogenated (4% Pd/C) at 50 psig. Thereduced product was adsorbed onto Dowex 50X8 (acid form), washed andeluted with NH₄ OH-methanol. After evaporation of the solvents in vacuo,the residual oil was dissolved in water and lyophilized. GC-mass specafter acetylation indicated the presence of M+H=302, consistent with2-hydroxymethyl-(3R-trans)-3,4-pyrrolidinediol tetraacetate.

Example 8

0.47 Gram of N-butyl-1-deoxy-1-ribosamine was dissolved in 45 mL waterand the pH adjusted to 5.5 with HCl. This solution was filtered througha 0.2μ filter and placed in a sterile 500 mL shake flask. To this wereadded freshly thawed Gluconobacter oxydans cells to give approximately40 mg/mL (wet cell weight). The shake flask containing the suspensionwas rotated at 120 rpm, room temperature for 24 hours. At this point thesuspension was clarified by centrifugation and filtered through a 0.45μfilter, treated with 1.5 gram of charcoal and catalytically hydrogenatedin the presence of 1 gram 4% Pd/C. The hydrogenation product wasadsorbed onto Dowex 50X8 (H⁺ form), then eluted with NH₄ OH-methanol.After evaporation of the solvents in vacuo, the resulting oil wasdissolved in water, the pH adjusted to 6 with HCl and freeze-dried.After acetylation, GC-mass spec indicated the presence of a molecularion (M+H) at 316, consistent with1-(n-butyl)-2-hydroxymethyl-(3R-cis)-3,4-pyrrolidinediol triacetate.

Example 9

1 Gram of N-benzyl-1-deoxy-1-erythrosamine was dissolved in 50 mL waterand the pH adjusted from 10.23 to 5.21 with HCl. To this solution wereadded 2 grams Gluconobacter oxydans cells and the suspension shaken at120 rpm, and room temperature. The pH was readjusted to 4.8 to 5.3 asneeded. After 24 hours the cells were removed by centrifugation and thesupernatant recharged with another 2 grams Gluconobacter oxydans cells.After 72 hours the cells were again removed by centrifugation and thesupernatant frozen. GC analysis indicated the presence of at least 80%bioconversion of the 1-deoxy-1-N-benzylerythrosamine. After thawing, theyellow supernatant was treated with 1.2 grams activated charcoal (40minutes) and filtered. The colorless filtrate was adjusted to pH 10.0with NaOH and hydrogenated with 1 gram 4% Pd/C overnight. This was thenfiltered to remove catalyst. On standing, crystals formed which werefiltered off. After drying the filtrate, acetylation of a portion gatem/e 230 by GC-mass spec, consistent with the triacetate of2-hydroxymethyl-3(S)-hydroxyazetidine.

Example 10

1.8 Grams of N-butyl-1-deoxy-1-erythrosamine were dissolved in 4 mLwater, of which 2 mL were diluted to 50 mL with water; the pH was 5.25.This solution was filtered through a 0.2μ filter and placed in sterile500 mL shake flask. To this were added freshly thawed Gluconobacteroxydans cells to give approximately 40 mg/mL (wet cell weight). Theshake flask containing the suspension was rotated at 120 rpm, roomtemperature for 48 hours. GC analysis of acetylated samples of theclarified suspension indicated the presence of a modified derivative ofthe 1-deoxy-1-N-(n-butyl)erythrosamine.

Example 11

This example illustrates the use of a cell free extract of Gluconobacteroxydans. 61 grams of Gluconobacter oxydans cell paste was suspended in108 grams of water. The cells were disrupted by three passes through aFrench press at 20,000 psig, giving greater than 95% disruption asdetermined by microscopic examination. The homogenate was centrifuged at43,000XG, 2° C., for 3 hours. The supernatant was carefully decantedyielding a cell free extract. The pellet contained the cell fragments.To 50 mL of N-butylmannosamine at approximately 50 grams per liter(assay by HPLC indicated 49 gm/L as the hydrochloride salt) in a 500 mLshake flask were added 3.5 grams of the cell free extract. The shakeflask was then rotated at 120 rpm at room temperature for 16 hours, atwhich time HPLC assay indicated that 39% of theN-butyl-1-deoxy-1-mannosamine had been converted to the corresponding6-butylamino-6-deoxy-D-fructofuranose.

Example 12

To 50 mL of N-butyl-1-deoxy-1-mannosamine at approximately 50 grams perliter (assay by HPLC indicated 49 gm/L as the hydrochloride salt) in a500 mL shake flask were added 3.5 grams of the resuspended cellfragments prepared in Example 11. This was rotated at 120 rpm at roomtemperature for 16 hours, at which time HPLC assay indicated that 99% ofthe N-butyl-1-deoxy-1-mannosamine had been converted to thecorresponding 6-(n-butyl)-amino-6-deoxy-D-fructofuranose.

Example 13

2.5 grams of N-butyl-1-deoxy-1-mannosamine were dissolved in 49 mL waterand the pH adjusted to less than 6. This solution was placed in a 500 mLshake flask and 2 grams G. oxydans cell paste were added. The shakeflask containing the suspension was shaken at 120 rpm at roomtemperature. After 24 hours, HPLC indicated that all of theN-butyl-1-deoxy-1-mannosamine had been converted to the corresponding6-butylamino-6-deoxy-D-fructofuranose. Cells were removed bycentrifugation and the supernatant treated with charcoal. The filtratefrom the charcoal treatment was diluted with 50 mL water andhydrogenated at 50 psi with 2 grams palladium on charcoal for 4 hours atroom temperature. After filtering off the catalyst, HPLC indicatedreduction to the 1-butyl-2-hydroxymethyl-3,4,5-piperidinetriol.

Example 14

2.8 grams of N-(2-naphthylmethyl)-1-deoxy-1-arabinosamine were dissolvedin 50mL water and the pH adjusted to 5 with hydrochloric acid. Thissolution was placed in a 500 mL shake flask and 2 grams G. oxyans cellpaste were added. The shake flask containing the suspension was shakenat 120 rpm at room temperature. After 22 hours the cells were removed bycentrifugation. 15 mL of the supernatant was chilled in ice, adjusted topH 12 with sodium hydroxide diluted with 20 mL methanol and reduced withsodium borohydride. After reduction at refrigeration temperatures thesolution was acidified and lyophilized. After acetylation of a sample ofthe dry powder, GC-mass spec gave m/e 400 (M+H), corresponding to1-(2-naphthylmethyl)-2-hydroxymethyl-(3R-cis)-3,4-pyrrolidinediol.

Example 15

This example demonstrates the preparation ofN-benzyl-1-deoxy-1-erythrosamine. In a Parr reactor, four grams oferythrose were suspended in 10 mL of benzylamine, 50 mL water and 50 mLmethanol. To this suspension was added 5 grams of Raney nickel and themixture hydrogenated at 50 psi and 50° C. for four hours. the catalystwas filtered off and rinsed with 150 mL water which was combined withthe filtrate. The total filtrate was acidified an treated with anionexchange resin. The product was eluted from the resin withmethanol-aqueous ammonia, the solvents evaporated in vacuo and theresulting recrystallized from ethanol-ethyl acetate-ether.

Example 16

This example demonstrates the preparation ofN-(4-picolinyl)-1-deoxy-1-mannosamine. 15.5 grams of D(+)-mannose weresuspended in 9.5 mL 4-(aminomethyl)pyridine and 200 mL methanol. To thissuspension were added 5 grams Raney nickel and the mixture hydrogenatedat 60 psi and 50° C. for 6.5 hours. The catalyst was filtered off andthe solvents evaporated in vacuo. The resulting oil was crystallizedfrom ethanol.

Example 17

This example demonstrates the preparation ofN-(4-picolinyl)-1-deoxy-1-arabinosamine. 15.5 grams of D(-)-arabinosewere dissolved in 9.5 mL 4-(aminomethyl)pyridine, 135 mL methanol and 65mL water. To this solution were added 5 grams Raney nickel and themixture hydrogenated at 60 psi and 50° C. for 6.25 hours. The catalystwas filtered off and the solvents evaporated in vacuo. The resulting oilwas crystallized from ethanol.

Although the invention has been described with respect to specificmodifications, the details thereof are not to be construed aslimitations, for it will be apparent that various equivalents, changesand modifications may be resorted to without departing from the spiritand scope thereof and it is understood that such equivalent embodimentsare to be included therein.

That which is claimed is:
 1. A process for producing a compound selectedfrom the group consisting of N-substituted polyhydroxy piperidines basedon N-substituted mannosamines, allosamines and altrosamines,N-substituted polyhydroxy pyrrolidines, N-substituted polyhydroxyazetidines, and salts thereof, comprising:(a) contacting, underconditions suitable for microbial oxidation, (i) an N-substituted aminosugar compound selected from the group consisting ofN-substituted-1-deoxy-1-hexosamines, based on mannose, allose andaltrose, N-substituted-1-deoxy-1-pentosamines,N-substituted-1-deoxy-1-tetrosamines, and salts thereof, wherein theN-substituted amino sugar compound lacks a nitrogen protecting group;and (ii) microorganisms or a fraction thereof selected from a groupconsisting of Acetobacteraceae and Corynebacterium, wherein themicroorganism or fraction thereof is capable of oxidizing theN-substituted amino sugar compound, to produce a corresponding oxidizedcompound selected from the group consisting ofN-substituted-amino-6-deoxy-2-ketohexuloses based on mannose, allose andaltrose, N-substituted-amino-5-deoxy-2-ketopentuloses,4-(N-substituted)-amino-1,3-dihydroxy-2-butanones and salts thereof; and(b) reducing the oxidized compound produced in step (a) to produce acorresponding compound selected from the group consisting ofN-substituted polyhydroxy piperidines based on N-substitutedmannosamines, allosamines and altrosamines, N-substituted polyhydroxypyrrolidines, N-substituted polyhydroxy azetidines, or salt thereof. 2.The process according to claim 1 wherein the substituent on the nitrogenis selected from the group consisting of phenyl, C₁ -C₁₀ alkyl, C₁ -C₁₀alkyl substituted with aromatic, amide or carboxy, and C₂ -C₁₀ alkylsubstituted with hydroxy.
 3. The process according to claim 2 whereinN-benzyl-1-deoxy-1-mannosamine is oxidized to produce6-benzylamino-6-deoxy-D-fructofuranose which is then reduced to produce1-benzyl-2-hydroxymethyl- 3R-(3α, 4β, 5β)!-3,4,5-piperidinetriol.
 4. Theprocess according to claim 2 wherein N-benzyl-1-deoxy-1-mannosamine isoxidized to produce 6-benzylamino-6-deoxy-D-fructofuranose which is thenreduced to produce 2-hydroxymethyl- 3R-(3α, 4β,5β)!-3,4,5-piperidinetriol.
 5. The process according to claim 2 whereinN-(2-naphthylmethyl)-1-deoxy-1-mannosamine is oxidized to produce6-(2-naphthylmethylamino)-6-deoxy-D-fructofuranose which is then reducedto produce 1-(2-naphthylmethyl)-2-hydroxymethyl- 3R-(3α, 4β,5β)!-3,4,5-piperidinetriol.
 6. The process according to claim 2 whereinN-(2-naphthylmethyl)-2-deoxy-1-mannosamine is oxidized to produce6-(2-naphthylmethylamino)-6-deoxy-D-fructofuranose which is then reducedto produce 2-hydroxymethyl- 3R-(3α, 4β, 5β)!-3,4,5-piperidinetriol. 7.The process according to claim 2 wherein N-butyl-1-deoxy-1-mannosamineis oxidized to produce 6-butylamino-6-deoxy-D-fructofuranose which isthen reduced to produce 1-(n-butyl)-2-hydroxymethyl- 3R-(3α, 4β,5β)!-3,4,5-piperidinetriol.
 8. The process according to claim 2 whereinN-butyl-1-deoxy-1-arabinosamine is oxidized to produce5-butylamino-5-deoxy-D-threo-2-pentulose which is then reduced toproduce 1-(n-butyl)-2-hydroxymethyl-(3R-trans)-3,4-pyrrolidinediol. 9.The process according to claim 2 whereinN-benzyl-1-deoxy-1-arabinosamine is oxidized to produce5-benzylamino-5-deoxy-D-threo-2-pentulose which is then reduced toproduce 2-hydroxymethyl-(3R-trans)-3,4-pyrrolidinediol.
 10. The processaccording to claim 2 wherein N-butyl-1-deoxy-1-ribosamine is oxidized toproduce 5-butylamino-5-deoxy-L-erythro-2-pentulose which is then reducedto produce 1-(n-butyl)-2-hydroxymethyl-(3R-cis)-3,4-pyrrolidinediol. 11.The process according to claim 2 whereinN-benzyl-1-deoxy-1-erythrosamine is oxidized to produce4-benzylamino-(S)-1,3-dihydroxy-2-butanone which is then reduced toproduce 2-hydroxymethyl-3(S)-hydroxyazetidine.
 12. The process accordingto claim 2 wherein N-butyl-1-deoxy-1-erythrosamine is oxidized toproduce 4-butylamino-(S)-1,3-dihydroxy-2-butanone which is then reducedto 1-(n-butyl)-2-hydroxymethyl-3(S)-hydroxyazetidine.
 13. The processaccording to claim 1 wherein said microorganism is selected from thegroup consisting of bacteria of the genus Gluconobacter and bacteria ofthe genus Acetobacter.
 14. The process according to claim 13 whereinsaid microorganism is Gluconobacter oxydans.
 15. The process accordingto claim 14 wherein said microorganism is Gluconobacter oxydans subsp.suboxydans.
 16. The process according to claim 1 wherein saidmicroorganism is Curtobacterium betae.
 17. The process according toclaim 1 wherein said microorganism is used in the form of a cellsuspension.
 18. The process according to claim 1 wherein saidmicroorganism is used in the form of immobilized cells.
 19. A processfor producing a compound selected from the group consisting ofN-substituted polyhydroxy piperidines based on N-substitutedmannosamines, allosamines and altrosamines, N-substituted polyhydroxypyrrolidines, N-substituted polyhydroxy azetidines, and salts thereof,comprising:(a) aminating a sugar selected from the group consisting ofmannose, allose altrose, ribose, arabinose and erythrose to produce acorresponding amino sugar compound selected from the group consisting ofN-substituted-1-deoxy-1-hexosamines, based on mannose, allose andaltrose, N-substituted-1-deoxy-1-pentosamines,N-substituted-1-deoxy-1-tetrosamines, and salts thereof; (b) contacting,under conditions suitable for microbial oxidation, (i) the N-substitutedamino sugar compound produced in step (a), wherein the N-substitutedamino sugar compound lacks a nitrogen protecting group, and (ii)microorganisms or a fraction thereof selected from a group consisting ofAcetobacteraceae and Corynebacterium, wherein the microorganism orfraction thereof is capable of oxidizing the N-substituted amino sugarcompound, to produce a corresponding oxidized compound selected from thegroup consisting of N-substituted-amino-6-deoxy-2-ketohexuloses based onmannose, allose and altrose,N-substituted-amino-5-deoxy-2-ketopentuloses,4-(N-substituted)-amino-1,3-dihydroxy-2-butanones and salts thereof; and(c) reducing the oxidized compound produced in step (b) to produce acorresponding compound selected from the group consisting ofN-substituted polyhydroxy piperidines based on N-substitutedmannosamines, allosamines and altrosamines, N-substituted polyhydroxypyrrolidines, N-substituted polyhydroxy azetidines, and salts thereof.20. The process according to claim 19 wherein the substituent on thenitrogen is selected from the group consisting of phenyl, C₁ -C₁₀ alkyl,C₁ -C₁₀ alkyl substituted with aromatic, amide or carboxy, and C₂ -C₁₀alkyl substituted with hydroxy.
 21. The process according to claim 20wherein mannose is aminated to N-benzyl-1-deoxy-1-mannosamine which isoxidized to produce 6-benzylamino-6-deoxy-D-fructofuranose which is thenreduced to produce 1-benzyl-2-hydroxymethyl- 3R-(3α, 4β,5β)!-3,4,5-piperidinetriol.
 22. The process according to claim 20wherein mannose is aminated to N-benzyl-1-deoxy-1-mannosamine which isoxidized to produce 6-benzylamino-6-deoxy-D-fructofuranose which is thenreduced to produce 2-hydroxymethyl- 3R-(3α, 4β,5β)!-3,4,5-piperidinetriol.
 23. The process according to claim 20wherein mannose is aminated toN-(2-naphthylmethyl)-1-deoxy-1-mannosamine which is oxidized to produce6-(2-naphthylmethylamino)-6-deoxy-D-fructofuranose which is then reducedto produce 1-(2-naphthylmethyl)-2-hydroxymethyl- 3R-(3α, 4β,5β)!-3,4,5-piperidinetriol.
 24. The process according to claim 20wherein mannose is aminated toN-(2-naphthylmethyl)-1-deoxy-1-mannosamine which is oxidized to produce6-(2-naphthylmethylamino)-6-deoxy-D-fructofuranose which is then reducedto produce 2-hydroxymethyl- 3R-(3α, 4β, 5β)!-3,4,5-piperidinetriol. 25.The process according to claim 20 wherein mannose is aminated toN-butyl-1-deoxy-1-mannosamine which is oxidized to produce6-butylamino-6-deoxy-D-fructofuranose which is then reduced to produce1-(n-butyl)-2-hydroxymethyl- 3R-(3α, 4β, 5β)!-3,4,5-piperidinetriol. 26.The process according to claim 20 wherein arabinose is aminated toN-butyl-1-deoxy-1-arabinosamine which is oxidized to produce5-butylamino-5-deoxy-D-threo-2-pentulose which is then reduced toproduce 1-(n-butyl)-2-hydroxymethyl-(3R-trans)-3,4-pyrrolidinediol. 27.The process according to claim 20 wherein arabinose is aminated toN-benzyl-1-deoxy-1-arabinosamine which is oxidized to produce5-benzylamino-5-deoxy-D-threo-2-pentulose which is then reduced toproduce 2-hydroxymethyl-(3R-trans)-3,4-pyrrolidinediol.
 28. The processaccording to claim 20 wherein ribose is aminated toN-butyl-1-deoxy-1-ribosamine which is oxidized to produce5-butylamino-5-deoxy-L-erythro-2-pentulose which is then reduced toproduce 1-(n-butyl)-2-hydroxymethyl-(3R-cis)-3,4-pyrrolidinediol. 29.The process according to claim 20 wherein erythrose is aminated toN-benzyl-1-deoxy-1-erythrosamine which is oxidized to produce4-benzylamino-(S)-1,3-dihydroxy-2-butanone which is then reduced toproduce 2-hydroxymethyl-3(S)-hydroxyazetidine.
 30. The process accordingto claim 20 wherein erythrose is aminated toN-butyl-1-deoxy-1-erythrosamine which is oxidized to produce4-butylamino-(S)-1,3-dihydroxy-2-butanone which is then reduced toproduce 1-(n-butyl)-2-hydroxymethyl-3(S)-hydroxyazetidine.
 31. Theprocess according to claim 19 wherein said microorganism is selectedfrom the group consisting of bacteria of the genus Gluconobacter andbacteria of the genus Acetobacter.
 32. The process according to claim 31wherein said microorganism is Gluconobacter oxydans.
 33. The processaccording to claim 32 wherein said microorganism is Gluconobacteroxydans subsp. suboxydans.
 34. The process according to claim 19 whereinsaid microorganism is Curtobacterium betae.
 35. The process according toclaim 19 wherein said microorganism is used in the form of a cellsuspension.
 36. The process according to claim 19 wherein saidmicroorganism is used in the form of immobilized cells.